Germ cells serve as a link between generations, producing gametes that propagate genetic material from parent to offspring. Understanding the mechanisms by which germ cells develop will help illuminate the nature of totipotency, the causes of infertility, and the origins of germ cell tumors. Much knowledge of germ cell development derives from studying model organisms that utilize cytoplasmic determinants to specify germ cell fate in early embryogenesis. In contrast, many organisms (including mammals) utilize inductive signals to specify germ cell fate later in development. In spite of these differences, germ cells specified by determinants and those formed inductively share several common intrinsic mechanisms, including transcriptional repression of somatic fates and post-transcriptional control of gene expression. Mechanistic studies of inductive germ cell development have been limited almost exclusively to mouse and important questions remain unanswered. For example, what transcriptional networks define and maintain germ cell fate? After a germ cell has been specified, what systemic factors link its further differentiation with te physiological status of the animal? The freshwater planarian, Schmidtea mediterranea, serves as a powerful model for studying these questions. It has prodigious regenerative abilities, based upon a population of adult stem cells that allow even tiny body fragments to regenerate complete individuals. In planarians, the germ cell lineage develops post- embryonically from the somatic stem cells; like the soma, the germ cell lineage can also be regenerated. Preliminary data generated in the applicant's laboratory have identified intrinsic and extrinsic regulators required for maintaining germ cells and for regulating their differentiation. Based on these data, two specific aims will be pursued: (i) to characterize the transcriptional network in early germ cells; and (ii) to dissect the neuropeptide-based systemic regulation of germ cell differentiation. Work performed under the first aim will use chromatin immuno-precipitation, next-generation sequencing, and computational approaches to identify targets of a germ cell-specific transcription factor required for maintaining early germ cells. Experiments conducted under aim two will characterize the neuropeptide receptor mediating the systemic regulation of germ cell development, identify the cell type(s) that express the receptor, and discover genes expressed in response to neuropeptide signaling. For both aims, the functional genomic tools for studying S. mediterranea (high- throughput in situ hybridization; RNA interference by feeding double-stranded RNA) will be used to validate target genes and determine their functions. These studies use a model organism with unique attributes, and take unbiased, genomic approaches to identify conserved factors; thus, they have great potential to innovate. This work is significan because of its potential: (i) to identify conserved genes required for proper germ cell development; and (ii) to provide a promising strategy for understanding and treating parasitic flatworms that infect hundreds of millions of people worldwide. PUBLIC HEALTH RELEVANCE: Germ cells give rise to the next generation by producing gametes (eggs and sperm). Studying how these cells are produced and regulated is relevant to understanding potential causes of infertility in humans and the cancers that result from inappropriate regulation of germ cells. This research uses the planarian to understand how germ cells develop because it is relatively easy to identify genes and study their functions in thi simple animal; analyzing genes shared between planarians and mammals will help us figure out how these genes function in mammalian germ cells.